Clathrin-mediated endocytosis (CME) occurs via the assembly of clathrin-coated pits (CCPs), specialized microdomains at the plasma membrane that undergo progressive maturation and eventually invaginate and pinch-off to form sealed clathrin-coated vesicles (CCV) carrying their cargo (receptor-ligand complexes) into the cell. In the past funding period we probed by quantitative live cell imaging the dynamics of CCP formation and maturation and tested the role(s) of actin assembly in CME. We developed image analysis software to track in an unbiased way every clathrin-coated structure (CCS) visible by total internal reflection fluorescence microscopy relative to actin cortex dynamics. Focusing first on the heterogeneous lifetimes of CCPs we identified by decomposition of the lifetime histogram two shorter-lived and one longer-lived sub-population, each by itself exhibiting a wide spectrum of lifetimes. Systematic RNAi and decomposition of the shifted lifetime distributions revealed endocytic accessory factors that affect either the relative contribution or the lifetime of one of the three subpopulations. From these data we derived a working model of the hierarchy of molecular events during CME, including the requirements for actin filaments to build a scaffold during the initiation of CCPs and to depolymerize and differentially repolymerize in a pit-size dependent fashion during internalization. Intriguingly, our data posit the existence of a restriction/checkpoint that ~20-30 s after initiation gates CCP progression to a productive CCS versus abortion. Our data suggests that checkpoint ris regulated by the GTPase dynamin and is sensitive to cargo concentration and CCP composition. Armed with this initial success in image-based analyses of CME, we propose in the next funding period to test the hypotheses that i) through interaction with early accessory factors of the endocytic machinery, dynamin serves to integrate multiple physical and chemical cues and to monitor the fidelity of CCP assembly;ii) the concentration and stoichiometry of cargo-specific adaptors are essential determinants of the efficiency of CCP maturation;and iii) that the molecular composition of CCPs is an essential determinant of both the efficiency and rate of CCP maturation. To conduct these studies we will develop new, highly sensitive and quantitative imaging assays to determine the molecular composition of individual CCPs. Next, we will devise deterministic and probabilistic mathematical models to integrate the data and to make molecularly explicit predictions of the interactions between endocytic accessory factors and their functions in the CME program. Our primary approach is to exploit the intrinsic heterogeneity in the wildtype composition and dynamics of CCPs as a source of information for modeling, avoiding the notorious distortions associated with molecular intervention studies. To accomplish this we will produce cell models, imaging protocols and data analysis tools that will raise the sensitivity of our experiments to a level where model predictions can also be validated by very mild perturbation experiments that do not affect the overall homeostasis of CME. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop quantitative imaging assays and mathematical models to decipher the regulation of the early stages of clathrin-mediate endocytosis at the cell surface. Understanding the mechanisms of initiation of endocytosis is critical to augmenting current therapeutic strategies that target the balance of cell surface receptors as a means of altering signal transduction in disease-related cell pathologies. Understanding the initiation of endocytosis is also critical to fight the proliferation of bacteria and viruses, which hijack the endocytic machinery to enter their host cells.